Propyl cationic peptide lipids, synthesis method thereof, and application thereof

Abstract

A class of propyl cationic peptide lipids is propyl cationic peptide lipid compounds having a general formula structure as follows. After the propyl cationic peptide lipids are dispersed in water, a cationic liposome with a particle size of approximately 100 nm is obtained. The cationic liposome can carry plasmid DNA (pDNA) or small interfering RNA (siRNA) into cells to realize the function of gene delivery, and is almost non-toxic to the cells.

The invention claimed is: 
     
       1. A propyl cationic peptide lipid having a structure of general formula I: 
       
         
           
           
               
               
           
         
         wherein, 
         R 1  is selected from C 1-20  alkyl, x is an integer from 1 to 6, and (AA) x  represents an amino acid selected from the group consisting of lysine (Lys), ornithine (Orn), arginine (Arg), histidine (His), aspartic acid (Asp), and alanine (Ala), or a peptide comprising Orn and at least one amino acid selected from the group consisting of Lys, Orm, Arg, His, Asp, Ala, and glycine (Gly). 
       
     
     
       2. The propyl cationic peptide lipid according to  claim 1 , wherein R 1  is selected from C 12 , C 14 , C 16 , or C 18  alkyl, x is selected from 2 to 4. 
     
     
       3. A method for synthesizing a propyl cationic peptide lipid, comprising the following steps:
 1) preparing a peptide head intermediate by protecting amino group of amino acid with a protective reagent by means of orthogonal protection method: the amino acid being selected from Lys, Orn, Arg, His, Asp, Ala or Gly, the amino group protective reagent being di-tert-butyl dicarbonate (Boc2O), Fluorenylmethoxycarbonyl succinimide (Fmoc-OSu) or benzyl chloroformate (CbzCl), at a molar ratio of the protective reagent and the amino acid being 1:1 to 8:1, the reaction solvent being acetonitrile, toluene, acetone, tetrahydrofuran or water, the reaction time being 1 to 20 hours, and the reaction temperature being 0 to 100° C., after the reaction, the solvent being removed by rotary evaporation, followed by purification by means of recrystallization with a recrystallization solvent being an ethyl acetate/petroleum ether mixed solvent (v/v=3:1); 
 2) protecting the amino group of 3-amino-1,2-propanediol with Fmoc-OSu protective reagent, after acylation with an acylating agent, reacting the resultant with alkyl amine, to prepare a disubstituted propyl long carbon chain intermediate: the acylating agent being carbonyl diimidazole, at a molar ratio of the acylating agent and 3-amino-1,2-propanediol being 1:1 to 3:1, after acylation, the molar ratio of 3-amino-1,2-propanediol to the alkyl amine being from 1:1 to 8:1, the reaction solvent being 30 ml to 300 ml of toluene, dichloromethane, DMF or chloroform, the reaction time being 12 to 48 hours, the reaction temperature being 25-100° C., after the reaction, the solvent being removed by rotary evaporation at 70° C., recrystallization being carried out with DMF, ethanol, ethyl acetate, water or ethanol/water mixed solvent; 
 3) linking the peptide head intermediate prepared in step (1) with the double long carbon chain intermediate prepared in step (2) via acylation:
 a. activating the peptide head intermediate firstly with an activating agent which is 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), N,N′-dicyclohexyl carbonimide (DCC) or 1-hydroxybenzotriazole (HOBt), at a molar ratio of the peptide head intermediate to the activating agent being 1:1 to 1:8, the reaction temperature being 0 to 60° C., the reaction time being 0.5 to 24 hours; 
 b. adding a solution of the double long carbon chain intermediate in dichloromethane, DMF or chloroform to the reaction solution in step a, after acylation, an amide bond being generated between the amino group of the intermediate and the carboxyl group of the peptide head intermediate, the peptide head intermediate reacting with the double long carbon chain intermediate at a molar ratio of 1:1 to 8:1, the reaction solvent being toluene, DMF, chloroform, acetone or methylene chloride, the reaction time being 12 to 96 hours, the reaction temperature being 20 to 100° C.; 
 
 4) removing the protective group with an amino de-protection agent which is 10% NaHCO 3  (w/v) or trifluoroacetic acid, at a molar ratio of the de-protection agent and a lipoid compound being 1:1 to 1:2, the de-protection time being 1 to 8 hours, the de-protection temperature being 0 to 4° C., the product being purified by recrystallization to obtain a crude product, the recrystallization solvent being ethyl acetate, acetonitrile, ethanol, water or anhydrous ether; 
 5) carrying out purification by column chromatography after the recrystallization, the crude product being dissolved in chloroform and purified with a silica gel column, followed by elution with a mixed solvent of methanol/chloroform (at a volume ratio of 3:1), the solvent being removed by rotary evaporation, followed by lyophilization, to obtain a cationic peptide lipid compound containing one amino acid head; 
 6) synthesizing other propyl cationic peptide lipid using the cationic peptide lipid compound containing one amino acid head as a raw material: subjecting the peptide head intermediate prepared in step (1) and the cationic peptide lipid containing one amino acid head prepared in step (5) to amino activation and acylation, to obtain a cationic peptide lipid compound of which the head is 1 to 6 amino acid(s), at a molar ratio of the two being 1:8 to 8:1, the specific reaction conditions and purification method being the same as steps (3), (4) and (5). 
 
     
     
       4. A cationic peptide liposome prepared from the propyl cationic peptide lipid according to  claim 1 , wherein the cationic peptide liposome is a homogeneous and stable liposome which is positively charged on the surface thereof and has a particle size of about 100 nm formed by dispersing the propyl cationic peptide lipid in an aqueous phase. 
     
     
       5. A method for preparing the propyl cationic peptide liposome, comprising the following steps:
 (1) dissolving a propyl cationic peptide lipid and an additive in chloroform or methanol according to a molar ratio of the two being 1:8 to 8:1, the additive being lecithin, sucrose esters, dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylcholine (DOPC) or cholesterol, wherein the propyl cationic peptide lipid has a structure of general formula I: 
 
       
         
           
           
               
               
           
         
         
           wherein R 1  is selected from C 1-20  alkyl, x is an integer from 1 to 6, and (AA) x  represents an amino acid selected from the group consisting of lysine (Lys), ornithine (Orn), arginine (Arg), histidine (His), aspartic acid (Asp), and alanine (Ala), or a peptide comprising Orn and at least one amino acid selected from the group consisting of Lys, Orm, Arg, His, Asp, Ala, and glycine (Gly); 
         
         (2) blowing the solution under nitrogen to form a uniform thin film, and drying in vacuum for 4 to 24 hours; 
         (3) performing hydration at 10 to 80° C. with ethanol, water or phosphate buffer for 1 to 10 hours, ultrasonic vibrating to transparent, to obtain the cationic peptide liposome with a concentration of 0.5 to 3 mg/ml. 
       
     
     
       6. A propyl cationic peptide liposome/gene complex prepared from the propyl cationic peptide liposome according to  claim 4 , wherein the propyl cationic peptide liposome according to  claim 4  and plasmid DNA (pDNA) or small interfering RNA (siRNA), by electrostatic interaction form homogeneous and stable nanoparticles dispersed in an aqueous phase. 
     
     
       7. A method for preparing the propyl cationic peptide liposome/gene complex according to  claim 6 , comprising the following steps:
 (1) taking 0.5 to 8 μl the propyl cationic peptide liposome according to  claim 4  and dispersing into 25 to 50 μl cell culture medium of DMEM or RPMI1640, mixing homogeneous to make a concentration being 0.02 μg/μ1 to 0.16 μg/μl; 
 (2) diluting pDNA or siRNA in the cell culture medium DMEM or RPMI1640, mixing homogeneous 0.5 to 1.0 μl to make a plasmid concentration being 0.02 μg/μl; 
 (3) mixing the two dilutions of (1) and (2) homogeneous according to a mass ratio between the liposome and gene of 1:1 to 8:1, placing at a room temperature for 10 to 40 min, to obtain the propyl cationic peptide liposome/gene complex. 
 
     
     
       8. A method for transecting a cell, the method comprising applying the propyl cationic peptide liposome/gene complex according to  claim 6  to the cell. 
     
     
       9. The propyl cationic peptide lipid according to  claim 1 , wherein (AA) x  is -Ala-Orn. 
     
     
       10. The propyl cationic peptide lipid according to  claim 1 , wherein (AA)x is -Orn-Lys-Lys. 
     
     
       11. The propyl cationic peptide lipid according to  claim 1 , wherein (AA) x  is -Ala-Orn-Lys-Lys.